The HIV/AIDS pandemic continues to wreak havoc on global scale and has become one of the most destructive pandemics in recorded history. Despite the tremendous improvements in life-extending antiretroviral therapy, it is likely that the only way by which the HIV/AIDS pandemic can be halted is through the development of an effective HIV vaccine. Unfortunately, conventional vaccines against HIV have provided little or no protection, highlighting the need for a better understanding of HIV-specific immune responses and for novel HIV vaccine approaches. Studies have demonstrated the important role of CD8+ T cells in controlling HIV infections, yet there is no clear consensus as to which viral proteins are responsible for eliciting effective CD8+ T cell responses. Particularly, information is lacking on the timing, magnitude and relevance of T cell- specific epitopes displayed by the infected CD4+ T cells during the course of HIV infection, yet these parameters are of crucial importance when considering specific HIV proteins as components in vaccine-based approaches or as targets for immunotherapy. The goal of the proposed research is to develop new reagents and methods, which can be performed in an ordinary laboratory setting, to assess the HIV antigen presentation profile on infected cells and relate these parameters with the temporal events occurring early during the course of HIV infection. We have been developing novel T cell receptor (TCR)-based reagents and simple methods to more directly quantitate and visualize peptide antigen presentation on diseased cells and tissues, including HIV-infected cells. We have shown that soluble single-chain TCR (scTCR) reagents can be used to evaluate endogenous antigen presentation on tumor cells and recently have extended these studies to generate high- affinity soluble scTCR reagents that are capable of recognizing HIV peptide epitopes as well as their known respective epitope variants. These reagents were found to be capable of recognizing HIV peptide/HLA complexes displayed on HIV-infected CD4 T cells by flow cytometry, verifying the feasibility of this approach for evaluating the HIV antigen presentation profile. Under this proposal, we intend to expand the reagent portfolio of HIV antigen recognition specificities and further optimize methods for analyzing antigen presentation levels and kinetics in HIV-infected T cells. The following specific aims will be conducted to achieve the goals of this project: 1) Generate and characterize a panel of soluble scTCR reagents specific for at least 20 different well-characterized HIV peptide/HLA complexes and 2) utilize these reagents to quantitate HIV peptide antigen presentation by HIV-infected cells. The results of these studies will help establish which specific HIV epitopes may play a role in generating protective CD8 T cell responses early in infection and thus should aid in the selection of the most relevant HIV targets for preventative vaccines, an ultimate goal of this project. In parallel, we will rapidly commercialize the HIV-specific TCR reagents as part of our existing research reagent portfolio to allow their use beyond the studies contemplated in this proposal. PUBLIC HEALTH RELEVANCE: Current information is lacking on the timing, magnitude and relevance of HIV antigens displayed by patient's T cells during the course of viral infection, yet these parameters are of crucial importance when considering specific HIV proteins as components in vaccine-based approaches. The goal of the proposed research is to develop approaches to assess the HIV antigen presentation profile on infected cells and relate these parameters with the temporal events occurring early during the course of HIV infection. Ultimately, these studies will help establish which specific HIV antigens play a role in generating protective CD8+ T cell responses early in infection and thus should aid in the selection of the most relevant HIV targets for preventative vaccines.